Immunochromatographic Strip Based on Tetrahedral DNA Immunoprobe for the Detection of Aflatoxin B1 in Rice Bran Oil

Aflatoxin B1 (AFB1), a widespread contaminant in food and feeds, poses a threat to the health of animals and humans. Consequently, it is significant to develop a rapid, precise and highly sensitive analytical method for the detection of AFB1. Herein, we developed an immunochromatographic strip (ICS) based on a tetrahedral DNA (TDN) immunoprobe for AFB1 determination in rice bran oil. Three sizes of TDN immunoprobes (AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, AuNP-TDN26bp-mAb) were constructed, and the performance of these three immunoprobes, including the effective antibody labeling density and immunoaffinity, was measured and compared with that of the immunoprobe (AuNP-mAb) developed using the physical adsorption method. Subsequently, the optimal TDN immunoprobe, namely AuNP-TDN13bp-mAb, was selected to prepare the immunochromatographic strip (ICS) for the qualitative and quantitative detection of AFB1 in rice bran oil. The visual limits of detection (vLODs) of the ICS based on AuNP-TDN13bp-mAb and AuNP-mAb were 0.2 ng/mL and 2 ng/mL, with scanning quantitative limits (sLOQs) of 0.13 ng/mL and 1.4 ng/mL, respectively. The ICS demonstrated a wide linear range from 0.02 ng/mL to 0.5 ng/mL, with good specificity, accuracy, precision, repeatability, and stability. Moreover, a high consistency was observed between the constructed ICS and ultra-high-performance liquid chromatography (UPLC) in the quantification of AFB1. The results indicated that the introduction of TDN was beneficial for promoting efficient antibody labeling, protecting the bioactivity of immunoprobes, and increasing the sensitivity of detection, which would provide new perspectives for the achievement of the highly sensitive detection of mycotoxins.


Introduction
Aflatoxins are toxic secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus [1,2].Among them, aflatoxin B 1 (AFB 1 ) is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC) due to its high toxicity [3].Excessive and prolonged exposure to AFB 1 can result in teratogenic, mutagenic, and carcinogenic diseases, posing a significant threat to animals and human beings [4].Furthermore, AFB 1 is widely present in agricultural products and feeds, whose contamination can occur in any procedure from farm to table [5].Rice bran oil, as a prevalent and nutritious vegetable oil, is rich in gluten, tocopherol, phytosterol, and other nutrients and is extracted from rice bran, which is susceptible to being contaminated with aflatoxins [6][7][8][9].Due to its intense toxicity and widespread distribution, many countries and regions have set maximum levels of AFB 1 .Hence, it is crucial to establish a rapid, efficient, and sensitive method for the detection of AFB 1 to ensure global food safety [10,11].
At present, one of the typical analytical approaches available for AFB 1 detection focuses on instrumental analysis, mainly including liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) [12][13][14].Despite their reliability and accuracy, these methods often require expensive equipment and technical personnel for precise quantification.In contrast, enzyme-linked immunosorbent assay (ELISA)-based detection methods have gained wide application due to their low-cost and straightforward operation [15,16].Nevertheless, the time-consuming incubation and washing procedures restrict their practical application.Therefore, it is imperative to establish a method for the rapid on-site detection of AFB 1 [17].
Compared with the aforementioned methods, the immunochromatographic strip (ICS) method, which is rapid, convenient, simple, and affordable, has been widely used in the detection of AFB 1 .However, the sensitivity of conventional ICSs based on colloidal gold nanoparticles (AuNPs) is relatively low [18].Researchers have exerted numerous efforts to improve the sensitivity of AuNP-based ICSs.Novel nanoparticles are receiving increased attention due to their large specific surface area and unique physical properties, which are capable of capturing more antibodies or amplifying the readout signal for improvements in their sensitivity [19][20][21].However, the orientation of the antibody on the solid-phase carrier is of significance in establishing a highly sensitive detection method since the ideal orientation of an antibody could fully expose the antigen-binding sites, giving rise to the intense recognition between antibodies and targets.To address this issue, tetrahedral DNA nanostructures (TDNs) were incorporated into the assembly process of the immune probe.
A TDN is a type of three-dimensional (3D) pyramid structure formed through the self-assembly of four complementary single-strand DNA (ssDNA) by annealing.Owing to its advantages such as simple synthesis, structural rigidity, and high programmability, it has been extensively employed in the field of biosensing [22][23][24].Lin et al. [25] developed a biosensor using TDN, which can precisely tune the anchoring density, by designing different sizes of TDNs [26][27][28][29][30]. Currently, TDNs are mostly used in electrochemical detection, primarily on two-dimensional electrodes.Inspired by these advancements, we endeavored to attach TDNs to 3D colloidal gold particles to accurately control the antibody density with the aim of significantly enhancing the target accessibility and the signal sensitivity [31][32][33][34][35].
In this study, three immunoprobes were constructed based on TDNs with different diameters.The distinctive characteristic of TDN was employed to improve the labeling density of the effective antibodies, protecting the antigen-binding fragment (Fab) from sheltering due to the disordered orientation of antibodies.Subsequently, the immunoprobe with the best performance was selected for the development of ICSs for the highly sensitive detection of AFB 1 in rice bran oil.It is worth noting that the introduction of TDN greatly facilitates the effective labeling of antibodies, which protects the bioactivity of the antibody, reduces the dosage of the antibody, and makes cost savings possible.More importantly, the site-directed immobilization of antibodies is conducive to the specific recognition of the antibody and target, which has the potential to be extended to the establishment of highly sensitive detection methods for other contaminants.

Self-Assembly of TDNs
TDNs were synthesized according to the previous literature [36].In brief, four DNA strands (A, B, C, D) were mixed in TM buffer to a final concentration of 1 µmol/L and reacted with TCEP.An equal amount of DNA was denatured at 95 • C for 10 min, followed by rapid annealing at 4 • C for 30 min to produce TDNs (TDN 13bp , TDN 17bp , TDN 26bp ).Then, the synthesized TDNs were characterized by poly acrylamide gel electrophoresis (PAGE) and the fluorescence resonance energy transfer (FRET).

Construction and Characterization of the Immunoprobes
The immunoprobes, namely AuNP-TDNs-mAb conjugates, were prepared as follows (Figure 1A): Optimal amounts of TDN 13bp , TDN 17bp , and TDN 26bp were dropped into 1 mL of AuNP and incubated at room temperature (RT) for at least 16 h to obtain the AuNP-TDNs (AuNP-TDN 13bp , AuNP-TDN 17bp , and AuNP-TDN 26bp ) conjugates.Upon stirring for 2 h, 50 µL of 10% BSA was added to seal the excess binding sites on the AuNP surfaces.Subsequently, the AuNP-TDNs conjugates were purified by centrifugation and resuspended in 1 mL of PBS.Then, 70 µL of the click chemical reaction liquid (0.12 g/mL AEPS) was added and allowed to react at RT for 3 h.Then, the optimal amount of mAb was added and incubated at RT for 2 h.Eventually, these three probes were purified by centrifugation and resuspended in 100 µL of running buffer (PBS containing 0.1% Tween-20 and 1% sucrose) for further use.Additionally, the immune probe (AuNP-mAb) based on physical adsorption labeling was prepared according to a previous report with minor modifications [37].To verify the successful construction of these immunoprobes, ultraviolet-visible absorption spectrum (UV-Vis), dynamic light scattering (DLS), a quartz crystal microbalance (QCM), and the zeta potential were utilized to characterize these three immunoprobes (AuNP-TDN 13bp -mAb, AuNP-TDN 17bp -mAb, AuNP-TDN 26bp -mAb).
was added and allowed to react at RT for 3 h.Then, the optimal amount of mAb was added and incubated at RT for 2 h.Eventually, these three probes were purified by centrifugation and resuspended in 100 µL of running buffer (PBS containing 0.1% Tween-20 and 1% sucrose) for further use.Additionally, the immune probe (AuNP-mAb) based on physical adsorption labeling was prepared according to a previous report with minor modifications [37].To verify the successful construction of these immunoprobes, ultraviolet-visible absorption spectrum (UV-Vis), dynamic light scattering (DLS), a quartz crystal microbalance (QCM), and the zeta potential were utilized to characterize these three immunoprobes (AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, AuNP-TDN26bp-mAb).

Estimation of the Labeling Density of Effective Antibody
The optimal amount of TDN was added to AuNP to obtain AuNP-TDN conjugates, followed by the addition of excess mAb, which were then incubated for 2 h at RT to acquire AuNP-TDN-mAb complexes.The coupling density of the antibody was calculated by determining the antibody in the supernatant before and after reaction using Coomassie bright blue.Thereafter, excess antigen was added and incubated along with the antibodies of the immunoprobe for 2 h at 37 °C, followed by the measurement of the unbound antigen via Coomassie bright blue.Ultimately, the labeling density of the effective antibody was estimated by measuring the consumption of the antigen captured by the antibody of the AuNP-TDN-mAb probe.The coupling density of the antibody and the labeling density of the effective antibody of AuNP-mAb were also estimated based on the above.

Investigation of the Immunoaffinity of the Immunoprobes
The immunoaffinity of these three AuNP-TNDs-mAb and AuNP-mAb immunoprobes was detected using ELISA.Briefly, a 96-well microplate was coated with AFB1-BSA (1 µg/mL, 100 µL/well) in CBS buffer and incubated overnight at 4 °C, followed by six washes with PBST (PBS containing 0.05% Tween-20).After washing, 200 µL of 1% BSA was added to each well, which was incubated for 2 h at 37 °C.Subsequently, a series of concentrations (0.01, 0.02, 0.05, 0.1, 0.2, 0.4, and 0.5 mg/mL) of each immunoprobe were added successively and incubated for another 1 h at 37 °C.Then, 100 µL of 3.2 µg/mL IgG-HRP was added and incubated for 1 h at 37 °C, followed by the addition of TMB (100 µL) as substrate.After 15 min of incubation, 100 µL of H2SO4 was added to terminate the reaction, and the absorbance of the mixture was measured immediately at 450 nm.The Hill equation curves of these four immunoprobes (AuNP-mAb, AuNP-TDN13bp-mAb, AuNP-

Estimation of the Labeling Density of Effective Antibody
The optimal amount of TDN was added to AuNP to obtain AuNP-TDN conjugates, followed by the addition of excess mAb, which were then incubated for 2 h at RT to acquire AuNP-TDN-mAb complexes.The coupling density of the antibody was calculated by determining the antibody in the supernatant before and after reaction using Coomassie bright blue.Thereafter, excess antigen was added and incubated along with the antibodies of the immunoprobe for 2 h at 37 • C, followed by the measurement of the unbound antigen via Coomassie bright blue.Ultimately, the labeling density of the effective antibody was estimated by measuring the consumption of the antigen captured by the antibody of the AuNP-TDN-mAb probe.The coupling density of the antibody and the labeling density of the effective antibody of AuNP-mAb were also estimated based on the above.

Investigation of the Immunoaffinity of the Immunoprobes
The immunoaffinity of these three AuNP-TNDs-mAb and AuNP-mAb immunoprobes was detected using ELISA.Briefly, a 96-well microplate was coated with AFB 1 -BSA (1 µg/mL, 100 µL/well) in CBS buffer and incubated overnight at 4 • C, followed by six washes with PBST (PBS containing 0.05% Tween-20).After washing, 200 µL of 1% BSA was added to each well, which was incubated for 2 h at 37 • C. Subsequently, a series of concentrations (0.01, 0.02, 0.05, 0.1, 0.2, 0.4, and 0.5 mg/mL) of each immunoprobe were added successively and incubated for another 1 h at 37 • C.Then, 100 µL of 3.2 µg/mL IgG-HRP was added and incubated for 1 h at 37 • C, followed by the addition of TMB (100 µL) as substrate.After 15 min of incubation, 100 µL of H 2 SO 4 was added to terminate the reaction, and the absorbance of the mixture was measured immediately at 450 nm.The Hill equation curves of these four immunoprobes (AuNP-mAb, AuNP-TDN 13bp -mAb, AuNP-TDN 17bp -mAb, and AuNP-TDN 26bp -mAb) were fitted, for which the dissociation equilibrium constant (K d ) values were utilized to evaluate the immunoaffinity [38].

Sample Pretreatment
Firstly, 5 g of rice bran oil sample was transferred into a 50 mL centrifuge tube and mixed with 20 mL of 70% methanol, followed by 20 min of vortexing.After centrifugation at 6000 rpm for 10 min, the supernatant was filtered through a 0.22 µm membrane for further use.

Fabrication of the ICS
The AuNP-TDN 13bp -mAb immunoprobe with the optimal performance was selected for the preparation of ICS (Figure 1B) and was compared to the ICS based on AuNP-mAb.The ICS consists of four parts, namely a polyvinyl chloride (PVC) baseplate, a nitrocellulose (NC) membrane, a sample pad, and an absorbent pad.The sample pads were blocked with PBS containing 0.05% (v/v) Tween-20, 1% BSA, and 2.5% (m/v) sucrose, followed by drying overnight at 37 • C.Then, 0.5 mg/mL of pAb (0.5 mg/mL) and the optimal amount of AFB 1 -BSA were sprayed onto the NC membrane as the control (C) line and test (T) line at a rate of 0.8 µL/cm.The distance between the C and T lines was set to 8 mm.Next, the sample and absorbent pad were sequentially attached to both sides of the PVC baseplate with a 2 mm overlap and then divided into strips with a width of 4.5 mm.
The dosage of AuNP-TDN 13bp -mAb and the spray concentrations of AFB 1 -BSA on T line were optimized based on the chessboard method.The spray concentration of AFB 1 -BSA was set to 0.3, 0.4, and 0.5 mg/mL, while the dosage of the AuNP-TDN 13bp -mAb probe was set to 6, 7, 8, 9, and 10 µL, respectively.Under optimal conditions, ICSs were used to detect AFB 1 standard solutions with concentrations of 0 and 10 ng/mL.To achieve the best detection performance, both the color development of the T line and the detection sensitivity were taken into consideration.

Qualitative and Quantitative Detection of AFB 1 by ICSs
The appropriate amount of AuNP-TDN 13bp -mAb probe was thoroughly mixed with the sample extraction of rice bran oil and fully blended to obtain a 100 µL solution.Then, the test strip was inserted into the mixture and incubated at 37 • C for 15 min for the qualitative detection of AFB 1 by observing the color of the T and C lines.The visual limit of detection (vLOD) was defined as the concentration at which the minimum T line was significantly shallower than that of the negative control.The cut-off value was the concentration at which the T line completely vanished.
Meanwhile, the grayscale values of the C (GSc) and T (GS T ) lines were recorded with a scanner and quantitatively analyzed using ImageJ 2.12 software .The GS T /GS C values of the negative control and spiked samples were defined as GS 0 and GS, respectively.A series of spiked rice bran oil with final AFB 1 concentrations of 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, and 2 ng/mL were analyzed to establish the standard curve for the quantitative analysis.Particularly, the scanning limit of quantification (sLOQ) was defined as the concentration of AFB 1 corresponding to the value of the GS 0 mean plus the 10-fold standard deviation (SD) of the negative samples.Additionally, the spiked rice bran oil samples were simultaneously detected by this proposed immunosensor and ultra-high-performance liquid chromatography (UPLC), and then the t-test was conducted for significance analysis [39].

Characterization of the TDNs
The successful preparation of TDNs (TDN 13bp , TDN 17bp , and TDN 26bp ) was crucial for the subsequent construction of immunoprobes.Hence, 10% native PAGE was employed to characterize the stepwise synthesis of the TDNs with diverse sizes.As depicted in Figure 2A-C, the single B chain possessing a low molecular weight demonstrated the fastest mobility.With the synthesis of the TDNs, the spatial complexity as well as the molecular weight gradually increased, hindering the movement of the DNA strands.Consequently, the migration velocity of the BD and ABD hybrid chains was lower than that of the B chain.When the A, B, C, and D chains were incubated together, the slowest mobility emerged due to the formation of a TDN.To further certify the integrity of the TDNs, the B and D strands were, respectively, modified with fluorophore and quencher to investigate the FRET efficiency.As shown in Figure 3A-C, with the synthesis of TDNs, the FRET enhanced since the distance between the quencher cy5 and fluorophore cy3 decreased.In conclusion, TDNs with diverse sizes were successfully synthesized.
mobility.With the synthesis of the TDNs, the spatial complexity as well as the molecular weight gradually increased, hindering the movement of the DNA strands.Consequently, the migration velocity of the BD and ABD hybrid chains was lower than that of the B chain.When the A, B, C, and D chains were incubated together, the slowest mobility emerged due to the formation of a TDN.To further certify the integrity of the TDNs, the B and D strands were, respectively, modified with fluorophore and quencher to investigate the FRET efficiency.As shown in Figure 3A-C, with the synthesis of TDNs, the FRET enhanced since the distance between the quencher cy5 and fluorophore cy3 decreased.In conclusion, TDNs with diverse sizes were successfully synthesized.

Characterization of Immunoprobes
UV-vis absorption spectroscopy was carried out to confirm the successful preparation of these three AuNP-TDNs-mAb composites.In Figure 4A, a characteristic absorption peak of AuNP is presented at 520 nm.With the successive modification of the TDNs and mAb on the AuNP surfaces, the wavelength of the maximum adsorption displayed obvious redshifts, giving rise to the successful synthesis of AuNP-TDNs and AuNP-TDNs-mAb.In the DLS characterization (Figure 4B), the diameters of AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, and AuNP-TDN26bp-mAb increased from 18.75 nm to 28.05, 29.45, 32.28 nm, and then to 39.25, 42.25, and 44.31 nm, respectively.The estimated values were quite close to the theoretical values both for the TND diameter and for mAb with the ideal orientation.Conversely, the QCM frequency of these three probes decreased due to the increased mass after binding with the TDN and mAb (Figure 4C).Furthermore, the zeta potential showed a significant increase from −24 to about −9 mv for AuNP-TDNs-mAb (Figure 4D), demonstrating the successful synthesis of immunoprobes.mobility.With the synthesis of the TDNs, the spatial complexity as well as the molecular weight gradually increased, hindering the movement of the DNA strands.Consequently, the migration velocity of the BD and ABD hybrid chains was lower than that of the B chain.When the A, B, C, and D chains were incubated together, the slowest mobility emerged due to the formation of a TDN.To further certify the integrity of the TDNs, the B and D strands were, respectively, modified with fluorophore and quencher to investigate the FRET efficiency.As shown in Figure 3A-C, with the synthesis of TDNs, the FRET enhanced since the distance between the quencher cy5 and fluorophore cy3 decreased.In conclusion, TDNs with diverse sizes were successfully synthesized.

Characterization of Immunoprobes
UV-vis absorption spectroscopy was carried out to confirm the successful preparation of these three AuNP-TDNs-mAb composites.In Figure 4A, a characteristic absorption peak of AuNP is presented at 520 nm.With the successive modification of the TDNs and mAb on the AuNP surfaces, the wavelength of the maximum adsorption displayed obvious redshifts, giving rise to the successful synthesis of AuNP-TDNs and AuNP-TDNs-mAb.In the DLS characterization (Figure 4B), the diameters of AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, and AuNP-TDN26bp-mAb increased from 18.75 nm to 28.05, 29.45, 32.28 nm, and then to 39.25, 42.25, and 44.31 nm, respectively.The estimated values were quite close to the theoretical values both for the TND diameter and for mAb with the ideal orientation.Conversely, the QCM frequency of these three probes decreased due to the increased mass after binding with the TDN and mAb (Figure 4C).Furthermore, the zeta potential showed a significant increase from −24 to about −9 mv for AuNP-TDNs-mAb (Figure 4D), demonstrating the successful synthesis of immunoprobes.

Characterization of Immunoprobes
UV-vis absorption spectroscopy was carried out to confirm the successful preparation of these three AuNP-TDNs-mAb composites.In Figure 4A, a characteristic absorption peak of AuNP is presented at 520 nm.With the successive modification of the TDNs and mAb on the AuNP surfaces, the wavelength of the maximum adsorption displayed obvious redshifts, giving rise to the successful synthesis of AuNP-TDNs and AuNP-TDNs-mAb.In the DLS characterization (Figure 4B), the diameters of AuNP-TDN 13bp -mAb, AuNP-TDN 17bp -mAb, and AuNP-TDN 26bp -mAb increased from 18.75 nm to 28.05, 29.45, 32.28 nm, and then to 39.25, 42.25, and 44.31 nm, respectively.The estimated values were quite close to the theoretical values both for the TND diameter and for mAb with the ideal orientation.Conversely, the QCM frequency of these three probes decreased due to the increased mass after binding with the TDN and mAb (Figure 4C).Furthermore, the zeta potential showed a significant increase from −24 to about −9 mv for AuNP-TDNs-mAb (Figure 4D), demonstrating the successful synthesis of immunoprobes.

Determination of the Labeling Density of the Effective Antibody
Upon the addition of the appropriate TDNs (Figure S1) and excessive mAb, AuNP-TDNs-mAb was produced.To evaluate the performance of these three immunoprobes, the antibody coupling density and the effective antibody density were determined and compared to those of AuNP-mAb.As shown in Table 1, the antibody coupling densities of AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, AuNP-TDN26bp-mAb, and AuNP-mAb were 19, 16, 8, and 31 per AuNP, while the effective antibody densities of these four immunoprobes were 18, 16, 8, and 10 per AuNP, respectively, with effective labeling rates of 94.7%, 100%, 100%, and 32.3%, correspondingly.Although the AuNP-TDNs-mAb captured fewer antibodies, they exhibited high proportion of effective antibodies.This implies that the antibodies of AuNP-TDNs-mAb are beneficial to the extensive exposure of Fab, which are prone to ideal head-on orientation to improve the effective labeling of antibodies and further enhance the recognition between antibody and analyte.

Determination of the Labeling Density of the Effective Antibody
Upon the addition of the appropriate TDNs (Figure S1) and excessive mAb, AuNP-TDNs-mAb was produced.To evaluate the performance of these three immunoprobes, the antibody coupling density and the effective antibody density were determined and compared to those of AuNP-mAb.As shown in Table 1, the antibody coupling densities of AuNP-TDN 13bp -mAb, AuNP-TDN 17bp -mAb, AuNP-TDN 26bp -mAb, and AuNP-mAb were 19, 16, 8, and 31 per AuNP, while the effective antibody densities of these four immunoprobes were 18, 16, 8, and 10 per AuNP, respectively, with effective labeling rates of 94.7%, 100%, 100%, and 32.3%, correspondingly.Although the AuNP-TDNs-mAb captured fewer antibodies, they exhibited high proportion of effective antibodies.This implies that the antibodies of AuNP-TDNs-mAb are beneficial to the extensive exposure of Fab, which are prone to ideal head-on orientation to improve the effective labeling of antibodies and further enhance the recognition between antibody and analyte.

Investigation of the Immunoaffinity
The Hill equation was fitted and utilized to evaluate the immunoaffinity of the immunoprobes via the K d value, which are negatively correlated.As illustrated in Figure 5, the K d values of the AuNP-mAb, AuNP-TDN 13bp -mAb, AuNP-TDN 17bp -mAb, and AuNP-TDN 26bp -mAb immune probes were 6.2, 0.12, 0.25, and 1.6, respectively, indicating that the AuNP-TDNs-mAb immunoprobes exhibited stronger binding than AuNP-mAb.The introduction of TDN facilitated the specific recognition between antibodies and antigens.Considering the immunoaffinity and the effective labeling density of the antibody, the AuNP-TDN 13bp -mAb immunoprobe, due to its excellent performance, was selected for the subsequent ICS experiments.

Investigation of the Immunoaffinity
The Hill equation was fitted and utilized to evaluate the immunoaffinity of the immunoprobes via the Kd value, which are negatively correlated.As illustrated in Figure 5, the Kd values of the AuNP-mAb, AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, and AuNP-TDN26bp-mAb immune probes were 6.2, 0.12, 0.25, and 1.6, respectively, indicating that the AuNP-TDNs-mAb immunoprobes exhibited stronger binding than AuNP-mAb.The introduction of TDN facilitated the specific recognition between antibodies and antigens.Considering the immunoaffinity and the effective labeling density of the antibody, the AuNP-TDN13bp-mAb immunoprobe, due to its excellent performance, was selected for the subsequent ICS experiments.

AFB1 Analysis of the Proposed ICS
A total of 9 µL of AuNP-TDN13bp-mAb was mixed with 91 µL of AFB1-spiked sample extract.The mixture was analyzed by means of the proposed ICS, on which 0.3 mg/mL AFB1-BSA was sprayed on the T line.Under the optimal conditions, the vLOD of the ICS was 0.2 ng/mL with a cut-off value of 1 ng/mL (Figure 6A), which represents a significant improvement compared to that of the AuNP-mAb-based strip (vLOD, 2 ng/mL; cut-off value, 10 ng/mL).Moreover, the GS values of the C and T lines were measured by ImageJ software for a quantitative analysis.As shown in Figure 6B, the linear range of the quantification for the fabricated ICS was from 0.02 to 0.5 ng/mL.Correspondingly, the equation of the standard curve was y = −1.608x+ 1.161, with a correlation coefficient (R 2 ) of 0.993.The sLOQ of the ICS based on the AuNP-TDN13bp-mAb was 0.13 ng/mL, which is an increase of one order of magnitude compared to that of the ICS based on the AuNP-mAb probe (1.4 ng/mL).

AFB 1 Analysis of the Proposed ICS
A total of 9 µL of AuNP-TDN 13bp -mAb was mixed with 91 µL of AFB 1 -spiked sample extract.The mixture was analyzed by means of the proposed ICS, on which 0.3 mg/mL AFB 1 -BSA was sprayed on the T line.Under the optimal conditions, the vLOD of the ICS was 0.2 ng/mL with a cut-off value of 1 ng/mL (Figure 6A), which represents a significant improvement compared to that of the AuNP-mAb-based strip (vLOD, 2 ng/mL; cut-off value, 10 ng/mL).Moreover, the GS values of the C and T lines were measured by ImageJ software for a quantitative analysis.As shown in Figure 6B, the linear range of the quantification for the fabricated ICS was from 0.02 to 0.5 ng/mL.Correspondingly, the equation of the standard curve was y = −1.608x+ 1.161, with a correlation coefficient (R 2 ) of 0.993.The sLOQ of the ICS based on the AuNP-TDN 13bp -mAb was 0.13 ng/mL, which is an increase of one order of magnitude compared to that of the ICS based on the AuNP-mAb probe (1.4 ng/mL).

Assessment of the ICS
The specificity was evaluated by simultaneously detecting target mycotoxin AFB1 and four common non-target mycotoxins, namely OTA, ZEN, FB1, and T-2.As indicated in Figure 7, AFB1 could be specifically identified by the ICS.More importantly, the exist-

Assessment of the ICS
The specificity was evaluated by simultaneously detecting target mycotoxin AFB 1 and four common non-target mycotoxins, namely OTA, ZEN, FB 1 , and T-2.As indicated in Figure 7, AFB 1 could be specifically identified by the ICS.More importantly, the existence of all other mycotoxins, whether together or in isolation, did not have an impact on the response to AFB 1 .Only an evident red band at the T line was observed for the detection of AFB 1 , indicating the high selectivity of this proposed method.
tect AFB1 at concentrations of 0.05, 0.1, 0.3, 1, and 2 ng/mL.Only one false negative was observed, perhaps because the concentration of AFB1 was close to the vLOD, so was prone to being overlooked with the naked eye.
Meanwhile, the intra-assay and inter-assay precision of this proposed ICS was evaluated via recovery experiments.The results (Table 3) shows that the intra-assay and interassay recoveries ranged from 85.0% to 98.2% with a coefficient of variation (CV) varying from 5.23% to 7.47%, indicating acceptable accuracy and precision for the rapid quantitative screening of AFB1.
Furthermore, this developed ICS was compared with other immunochromatographic assays for the detection of AFB1.As shown in Table 4, the proposed ICS demonstrated a lower VLOD, good specificity, accuracy, and acceptable repeatability.Particularly, when compared with the random labeling of antibodies, the sensitivity was improved by the introduction of TDN, which could promote the specific recognition of the antibody and the target aflatoxin.In the repeatability experiment (Table 2), ICSs from the same batch were used to detect AFB 1 at concentrations of 0.05, 0.1, 0.3, 1, and 2 ng/mL.Only one false negative was observed, perhaps because the concentration of AFB 1 was close to the vLOD, so was prone to being overlooked with the naked eye.Meanwhile, the intra-assay and inter-assay precision of this proposed ICS was evaluated via recovery experiments.The results (Table 3) shows that the intra-assay and inter-assay recoveries ranged from 85.0% to 98.2% with a coefficient of variation (CV) varying from 5.23% to 7.47%, indicating acceptable accuracy and precision for the rapid quantitative screening of AFB 1 .Furthermore, this developed ICS was compared with other immunochromatographic assays for the detection of AFB 1 .As shown in Table 4, the proposed ICS demonstrated a lower V LOD, good specificity, accuracy, and acceptable repeatability.Particularly, when compared with the random labeling of antibodies, the sensitivity was improved by the introduction of TDN, which could promote the specific recognition of the antibody and the target aflatoxin.

Method Verification Using UPLC
Rice bran oil samples spiked with different concentrations of AFB 1 were used to evaluate the practicability of the ICS.As shown in Table 5, the results for the ICS and UPLC had no significant difference according to the t-test statistical method (|t| = 0.079, t α /2 = 2.132, |t| < t α /2).All these results illustrate that the ICS is applicable for the detection of AFB 1 in practical samples.

Conclusions
In this study, three novel immunoprobes based on TDNs with diverse diameters were constructed and applied for the fabrication of highly sensitive ICSs for the detection of AFB 1 in rice bran oil.With the introduction of TDNs, the effective antibody labeling density was extremely improved compared to that of the immunoprobe prepared via the adsorption labeling method, whose orientation is beneficial for fully exposing the Fab sites on the mAb.Considering the immunoaffinity and the effective labeling density of the antibody, AuNP-TDN 13bp -mAb, which showed excellent performance, was selected to fabricate the ICS for the qualification and quantification of AFB 1 .Under the optimal conditions, the vLOD was 0.2 ng/mL, and the sLOQ was 0.13 ng/mL, with a linear range of 0.02-0.5 ng/mL.Thanks

Figure 5 .
Figure 5. Hill equation curves and Kd values of probes.Kd: dissociation equilibrium constant.

Figure 5 .
Figure 5. Hill equation curves and K d values of probes.K d : dissociation equilibrium constant.

Table 1 .
The labeling density of the antibody.

Table 1 .
The labeling density of the antibody.

Table 2 .
Analysis of repeatability.
a Shows color; b color lightened obviously; c no color; abnormality rate % = (fn/20) × 100, fn is the number of repeats of abnormal color development of the detection line.

Table 3 .
The precision of the ICS method for AFB 1 detection in spiked rice bran oil samples.

Table 5 .
Comparison of the analytical results between the developed ICS and UPLC (n = 3).